The Experimental Study On Immortalized Human Neural Stem Cells Lineage By HTERT Gene Transfection

Neural stem cells (NSCs) possess the characteristics of multipotenty, self-duplication, self-renewal, and the potentiality of differentiating into various neural cells such as neuron, astrocyte and oligodendrocyte. NSCs have been found existing widely in the embryonic mammalian central nervous system (CNS) since Reynolds' founding of them in the mice striatum in 1990s, involving cerebral cortex, hippocampus, striatum, olfactory bulb, brain ventricle, diencephalons, midbrain, cerebellum, spinal cord, retina, and so on. NSCs also exist in subventricular zone of lateral ventricle and subgranular zone of hippocampus dentate gyrus in adult mammalian CNS.At present, it is easy to isolate, expand and culture NSCs in vitro. Induced by some factors, NSCs can differentiate into several nerve cell phenotypes.It has been confirmed that primary NSCs will divide and proliferate in vitro under the influence of mitogens like epidermal growth factor (EGF) and/or basic fibroblast growth factor (bFGF, FGF-2). Under such conditions, mouse-derived cells show a relatively rapid division rate (one per day), while human cells divide only once every 7-14 days, depending on the particular culture conditions. Followed by the telomere shortening, these cultured cells generally stop dividing at limited propagated numbers, which is named replicative senescence, thus possibly limiting their utility.Genetically propagated cell lines of human neural stem cells, because of their clonality, homogeneity, and possible faster division rate, may be better suitable to pursue certain types of cells for cellular and molecular studies than epigenetically expanded cell lines. Some scholars proposed the concept of immortalized neural stem cells, which is to halt the progression of developmental program by controlling the cells to remain in continuous cell cycle. Cells can be rendered immortal by a number of manipulations, although the most common method is to introduce neural stem cells with retroviral vectors encoding theoncogene and virus protein production. At present researchers have established several kinds of immortalized human neural stem cell lines by various methods, such as C10, JA3 cell lines which from embryo 13 weeks human hippocampus, H6 cell lines from embryo 15 weeks diencephalons and HNSC.100 cell lines from embryo 10.5 weeks diencephalons. The myc gene and SV40 large antigen are the most extensively used genes, all these cell lines were transfected by v-myc gene. Besides the normal neural stem cells features, the immortalized neural stem cells have the advantages as following: (1) They are self-renewing and can be expanded to large numbers in culture; (2) They will clearly enhance the efficiency of transgenesis procedures for rather easily generating numerous stable transduced cells in culture and expressing stable reporter and therapeutic genes; (3) They can be isolated as single clones; (4) The multipotent properties of the cells in vivo indicate that the immortalization process has not substantially altered their stem-cell or progenitor-like properties.The mechanism of cells immortalization is that the telomerase activity is activated when these cells transfected with oncogene. Telomerase catalytic subunit (hTERT) is the key factor of regulating telomerase activity. There were several reports on different somatic cells immortalization by hTERT gene.In our study, human neural stem cells from aborted human embryo cortex from 10 to 14 weeks gestational age were isolated, identified and expanded in vitro. Furthermore, we produced and established a cell lineage of the continuous immortalized neural stem cells by transfecting the neural stem cells with the PLXSN retroviral vector constructed with hTERT gene. The main methods and results are as follows:1. Isolated, identified and expanded the neural stem cells from 10 to 14 weeks gestational age aborted human embryo cortex in vitro.2. Amplification, purification and identification of retroviral vector PLXSN-hTERT.3. After PLXSN-hTERT packaged in PA317 cell lines, its expression in target cells were identified.4. The virus supernatant (contain retrovirus constructed with hTERT gene) could transfect the neural stem cells effectively. The telomerase activity was continuous high level. The hTERT mRNA was high level in transfected early stage, and then it decreased gradually and lasted in a low level. The hTERT mRNA wasn't parallel with telomerase activity.5. The immortalized neural stem cells kept undifferentiated feature and hadself-renewing capacity and multipotency. The speed of growth of the immortalized neural stem cells was obviously faster than the normal neural stem cells, and the division capacity was obviously strengthened.6. Transfected with hTERT gene, the immortalized neural stem cells which derived from human embryo possessed normal diploid and had no oncogenicity by chromosome analysis and nude mice transplanting experiment.In conclusion, we isolated, identified and expanded the neural stem cells from 10 to 14 weeks gestational age aborted human embryo cortex in vitro successfully, and transfected the neural stem cells with retrovirus which was constructed with hTERT gene. We confirmed that immortalized neural stem cells remain undifferentiated feature and had self-renewing capacity and multipotency. The speed of growth of the immortalized neural stem cells was obviously faster than the normal neural stem cells, and the division capacity was obviously strengthened. The immortalized neural stem cells could provide generous seed cells in further study.